Substance for lowering high cholesterol level in serum and methods for preparing and using the same

ABSTRACT

The invention relates to a substance which lowers LDL cholesterol levels in serum and which is fat soluble β-sitostanol fatty acid ester, and to a method for preparing and using the same. The substance can be taken orally as a food additive, food substitute or supplement. A daily consumption of the β-sitostanol ester in an amount between about 0.2 and about 20 g/day has been shown to reduce the absorption of biliary and endogenic cholesterol.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a Divisional Application which claims the benefits of pendingU.S. patent application Ser. No. 10/678,135, filed Oct. 6, 2003, nowU.S. Pat. No. 7,002,030 which is a Continuation of application Ser. No.10/289,341, filed Nov. 7, 2002 now U.S. Pat. No. 6,770,768, which is aDivisional of application Ser. No. 09/713,030 filed Nov. 16, 2000 nowU.S. Pat. No. 6,544,973, which is a Divisional of application Ser. No.09/190,598 filed Nov. 12, 1998, now U.S. Pat. No. 6,174,560 which is aDivisional of application Ser. No. 08/744,009 filed Nov. 5, 1996, nowU.S. Pat. No. 5,958,913 which is a Continuation-In-Part of applicationSer. No. 08/508,623, filed Jul. 28, 1995, now abandoned which is aContinuation-In-Part of application Ser. No. 08/140,085, filed Nov. 22,1993, now U.S. Pat. No. 5,502,045, which is a §371 of WO 92/19640, filedNov. 12, 1992, which claims priority from Finnish Patent ApplicationNumber PCT/FI91/00139, filed May 3, 1991. The disclosures of the priorapplications are hereby incorporated herein in their entirety byreference.

BACKGROUND OF THE INVENTION

A high cholesterol level in serum can be lowered effectively by alteringthe intestinal metabolism of lipids. In this case the aim may be tohamper the absorption of triglycerides, cholesterol or bile acids. Ithas been observed in a number of investigations that certain plantsterols, such as β-sitosterol (24-ethyl-5α-cholestane-3β-ol) and itshardened form, β-sitostanol (24-ethyl-5α-cholestane-3β-ol), lower serumcholesterol levels by reducing the absorption of dietary cholesterolfrom the intestines (1-25). The use of plant sterols can be consideredsafe, since plant sterols are natural components of vegetable fats andoils. Plant sterols themselves are not absorbed from the intestines, orthey are absorbed in very low concentrations. A decreased incidence ofcoronary disease is clearly associated with a decrease in serumcholesterol, in particular LDL cholesterol. A high serum cholesterolvalue is the most significant single indicator of the risk of coronarydisease.

The degree of cholesterol absorption depends on a hereditary property,apoprotein E-phenotype. Apoprotein E is a protein which belongs to serumlipoproteins and takes part in the transport of cholesterol in thesystem (26). Of alleles associated with the synthesis of apoprotein E,i.e. the lipoprotein which affects absorption, there are known threetypes, e2, e3, and e4, which combine in pairs at random. Alleles arecapable of forming in total six different combinations. The higher thesum of the subindices, the better absorbable the cholesterol and thehigher the level of cholesterol, in particular bad LDL cholesterol, inthe serum (27). e4 allele is over represented among the hereditaryfactors of Finns, so that its proportion is almost double as comparedwith many European populations (28).

Finns are indeed exceptionally sensitive to dietary flaws and to fattyand high-cholesterol food (29).

Serum cholesterol levels can be lowered by dietary means, by payingattention to the quantity and type of the fat ingested and to the amountof cholesterol intake. In practice, however, these means do not alwayslead to a satisfactory end result. Other methods, suitable for theentire population, for reaching serum cholesterol levels lower than thepresent ones must be searched for. Increasing the fiber content of foodis a method of limited effect. The cholesterol-lowering effect ofsoluble fiber in food is based on the binding and removal of bile acids.Since the absorption of cholesterol is of fundamental significance inthe regulation of the cholesterol level in serum, it is logical to aimat developing methods by which the absorption of cholesterol can beprevented or reduced.

Pollak demonstrated that ingested plant sterol lowered the level ofserum cholesterol in man (1). The same had previously been observed inexperimental animals (2, 3). It has been observed in a number ofinvestigations that large doses of plant sterols lower the levels ofserum cholesterol, at best by 10-20% (4, 5). In these experiments, largeamounts, up to 24 g/day, of β-sitosterol in crystalline form were used(6). In certain experiments the serum cholesterol level was loweredsignificantly even with lower doses (7), although a small amount ofsoluble sitosterol administered in the form of fatty acid esters did notseem to lower serum cholesterol very effectively (8). Sitosterolpreparations have in general been well tolerated in long-term use (9).

Natural plant sterols resemble cholesterol in their structure. Thedifferences between a cholesterol molecule and a plant sterol moleculeare primarily found in the structure of the side chain of the basicframe. An ordinary diet contains plant sterols 100-300 mg/day. Most ofthe plant sterol in the diet is β-sitosterol, and approximatelyone-third is campesterol. Small amounts of saturated 5α-sitostanols arealso present in food. Usually the campesterol concentrations in serum inparticular reflect the degree of absorption of cholesterol (10, 11, 12).Variation in the amounts of plant sterols in the diet affects the serumcholesterol level, but this is an area which has not been studied much.Plant sterols are poorly absorbed from the intestines. Plant sterolswhich are scantily absorbed into the system (less than 10% of thesterols) (30, 31, 32) are excreted in the bile and through that in thestools. At present it is easy to measure sterol levels from food, serumor stool samples by gas chromatographic methods. The levels in serum arein part dependent on the plant sterol amounts derived from the diet andin part on the efficiency of the absorption of sterols. In general theplant sterol levels in serum remain below 1/300 of the serum cholesterollevel, since the absorbed plant sterol fraction is excreted from thesystem in the bile.

Even large ingested doses of plant sterols do not show in serum plantsterol levels. The values remain at the normal level, since in man theplant sterol absorption capacity is rapidly saturated. The serum plantsterol level rises to a detrimental level in a few rare diseases such ascerebrotendinotic xanthomatosis and sitosterolemia (33, 34, 35), inconnection with which coronary disease is common. The incidence of thesediseases is at maximum a few cases in a population of one million. Not asingle case of these diseases has been observed in Finland. High plantsterol values are at times observed in patients suffering from certainhepatic diseases (36).

Studies of the metabolism of cholesterol have shown that sitosterolinhibits the absorption of both endogenic and dietary cholesterol fromthe intestines (13, 14). As a result of this, the excretion of neutralsteroids in the stools increases, which leads to a shortage ofcholesterol in the liver and through that to a decreased serumcholesterol level. On the other hand, sitosterol does not affect theabsorption of bile acids (13).

On the basis of experiments on animals, it seems that the action ofsitosterol is based on its ability to displace dietary cholesterol inbile acid micelli (15, 16, 17). Similar results have also been obtainedin man (37). Various plant sterols have been demonstrated to affect indifferent ways the absorption of cholesterol (19, 38). Previous studiescarried out on experimental animals give the impression that sitostanolis the most effective inhibitor of cholesterol absorption (38) and isitself almost nonabsorbable. Furthermore, an uncontrolled study on sixsubjects showed that free sitostanol (1.5 g/day) lowered the serumcholesterol (mainly LDL cholesterol) in four weeks by as much as 15%.During a pause of two weeks, the cholesterol values returned to theprevious levels (20). Most plant sterol preparations contain a number ofdifferent plant sterols. The effect of a plant sterol mixture on theabsorption of cholesterol varies, as does their own absorption (21, 22,23).

The studies carried out so far have mainly concentrated on investigatinghow the form (crystalline, suspension, granular) in which plant sterolsare dosed affects their efficacy in lowering serum cholesterol levels.Crystalline plant sterols do not to a significant degree dissolve in themicelli phase in the alimentary canal, and are therefore not capable ofefficiently inhibiting cholesterol absorption. Oils and fats are only toa limited degree capable of dissolving free sterols. Only in a dissolvedform do sterols inhibit the absorption of cholesterol. According toHeinemann and coworkers (24), sitostanol inhibited in an infusionexperiment the absorption of cholesterol 82%, whereas sitosterolinhibited the absorption 50%.

In certain studies, fatty acid esters of sitosterol, such as sitosterolacetate or oleate or stigmasterol oleate dissolved in fat, have beenused. In experiments on rats an “oil” of this type, having a sterolconcentration up to 8%, reduced the absorption of cholesterol by 20-40%(22). During a high-cholesterol diet (500 mg/day), sitosterol oleate (2g/day) dissolved in fat decreased the absorption of cholesterol in thetest subjects on average by 33% (25). In the same study, sitosterolmixed with food and in a lower dose (1 g/day) decreased the absorptionof cholesterol by 42%.

A German patent (Deutsches Patentamt, Offenlegungsschrift 2035069/Jan.28, 1971) relates to the adding of plant sterol fatty acid esters tocooking oil with the objective of lowering the serum cholesterol levelsin man. The said patent proposes for use in the esterification of freesterols a method which in no case fulfills the requirements for thepreparation of a food-grade product. According to the patent, theesterification is carried out between a free sterol and a fatty acidanhydride, with perchloric acid acting as a catalyst. The catalyst andreagent used cannot be accepted in a food process. In addition, the saidpatent relates to the fatty acid esters of only native plant sterols.

Many reagents which cannot be accepted as a food or for the productionof a product intended as an additive for foods have been used in thepreparation of sterol fatty acid esters. The use of, for example,chlorine (39), bromine (40), thionyl chloride (41) or anhydridederivatives of fatty acids is common. Of the methods previouslypatented, only the method of Baltes (Deutsches Patentamt,Offenlegungsschrift 2248921/Apr. 11, 1974) for the esterification ofsterols present in oils and fats by a chemical interesterificationtechnique fulfills the criteria of food processes. In the said patent,free sterol and an excess of fatty acid esters are added to a mixture ofoil or fat, whereafter the entire fatty mixture is interesterified by acommonly known interesterification technique.

BRIEF DESCRIPTION OF THE INVENTION

The present invention relates to a sterol of an entirely different typefor lowering the cholesterol levels in blood serum. The substancecomprises a fatty acid ester of 5∝ saturated sterols, especiallysitostanol fatty acid esters (sitostanol=24-ethyl-5∝-cholestane-3β-ol),which have been observed to lower cholesterol levels in serum withparticular efficacy.

The present invention includes a method of reducing the absorption ofcholesterol into the bloodstream from the digestive tract by orallyintroducing into the body an effective amount of a fatty acid ester of aβ-sitostanol. More preferably, the invention further includes orallyintroducing between about 0.2 and about 20 grams per day of β-sitostanolfatty acid ester into the body. The ester is introduced either as a foodadditive, a food substitute or a food supplement. When used as a foodadditive, the fatty acid ester of the β-sitostanol may be added to foodproducts such as cooking oils, margarines, butter, mayonnaise, saladdressings, shortenings, and other foods having an essential fatcomponent.

The invention includes a process for preparing a fat soluble substanceuseful for lowering the cholesterol levels in blood serum. The processcomprises the esterification of a stanol preferably β-sitostanol with afatty acid ester. The esterification is carried out in a food gradeprocess which is free of solvents. Typically, the fatty acid estercomprises a fatty acid or a mixture of fatty acids containing between 2and 22 carbon atoms. The esterification preferably is carried out at atemperature of 90°-120° C. under a vacuum of approximately 5-15 mmHgusing a catalyst such as sodium ethylate. The reaction is advantageouslycarried out between the free sitostanol and a fatty acid ester onlywithout the presence of other interesterifiable lipids (triglycerides orfats), resulting in a mixture of stanol fatty acid esters and fatty acidester.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the use of a sterol of an entirelydifferent type for lowering the cholesterol level in serum. What isinvolved is fatty acid esters of 5α-saturated sterols, especiallysitostanol fatty acid esters (sitostanol=24-ethyl-5α-cholestane-3β-ol)which have been observed to lower cholesterol levels in serum withparticular efficacy. The said esters can be prepared or used as such, orthey can be added to foods, especially to the fatty part of a food. Thesitostanol fatty acid ester mixture is prepared by hardening orhydrogenating a commercial β-sitosterol mixture(sitosterol=24-ethyl-5α-cholestane-3β-ol). β-sitostanol can be preparedby a prior known cholesterol hardening technique by hardeningβ-sitosterol by means of a Pd/C catalyst in an organic solvent (43). Ahardening degree of over 99% is achieved in the reaction. The catalystused in the hardening is removed by means of a membrane filter and theobtained sitostanol is crystallized, washed and dried. In accordancewith the invention, the β-sitostanol mixture, which contains campestanolapprox. 6%, is esterified with different fatty acid ester mixtures by acommonly known chemical interesterification technique (44, 45, 46). Amethyl ester mixture of the fatty acids of any vegetable oil can be usedin the reaction. One example is a mixture of rapeseed oil and methylester, but any fatty acids which contain approx. 2-22 carbon atoms areusable.

The method according to the invention for the preparation of stanolfatty acid esters deviates advantageously from the previously patentedmethods in that no substances other than free stanol, a fatty acid esteror a fatty acid ester mixture, and a catalyst are used in theesterification reaction. The catalyst used may be any knowninteresterification catalyst such as sodium ethylate.

It is also to be noted that in the method used in our application,contrary to the method of Baltes, referred to above, the fat itself isnot interesterified. In this case the fatty part of a fat preparation orsome other food will retain its natural properties. It should be notedfurther that the interesterified mixture can be added directly tofat-containing foods or be used as such. Since the stanol part of themixture is non-absorbable, the energy content of the stanol fatty acidester mixture is only 20-40% of the energy content of a conventional oilor fat, depending on the fatty acid composition. Thus the mixtures canbe used advantageously also as substances decreasing the energy contentor caloric value of a food.

The action of β-sitostanol fatty acid ester on cholesterol absorptionand on serum cholesterol levels has not been studied previously. Onestudy on which this application is based, investigated how plant sterolconcentrations in serum were affected by sitostanol (composition:β-sitostanol 94% and campestanol 6%), a hardened form of sitosterol,dissolved in rapeseed oil, both free and in the form of a fatty acidester. The test arrangement of the study is as follows:

Intervention Study TEST GROUPS (N-22)*-------------*----------------*---------------control (n-8)              ------------------*               β-sitostanol                                                (n = 7)              ----------------*----------------*β-sitostanol                                                ester                                                (n-7)*-------------*----------------*---------------* 0 wk.       6wks.           15 wks.         21 wks.INITIAL    EXPERIMENTAL     CONTINUATION PERIODThe first step for all groups was a rapeseed oil intervention (50 g/d),for the control group a rapeseed oil intervention for the duration ofthe test, and for the other groups a compound according to the testarrangement scheme, added to rapeseed oil.

Table 1 shows changes (%) caused during the experimental period in plantsterol levels in serum by β-sitostanol added to rapeseed oil, and duringthe continuation period with respect to β-sitostanol ester (3150 mg/d).It is noted that an increase in the β-sitostanol concentration of foodlowered the concentrations of both β-sitosterol and campesterol inserum, but did not produce a clear change in the serum β-sitostanolconcentrations.

TABLE 1 Stanol Added Change (%) Caused to Rapeseed by the Addition¹ Oil(mg/d) Campesterol β-sitosterol β-sitostanol β-sitostanol −18.4^(x)−13.0^(x) −0.6 (895) β-sitostanol −28.4^(x) −23.4^(x) −10.3 ester (895)²β-sitostanol −51.7^(x) −43.3^(x) −10.3 ester (3150)² ¹= Change in thetable has been corrected by the %-change in the control group which hadreceived rapeseed oil. ²= Amount in free stanol. ^(x)= Change issignificant as compared with the change in the control group, p < 0.05.The results also show that an intake of β-sitostanol in a solubleform—i.e. in the form of fatty acid esters reduced the absorption ofplant sterols more effectively than did free β-sitostanol taken in thesame dosage. With respect to fatty acid esters of β-sitostanols there isadditionally observed a clear dose response. It is evident thatβ-sitostanol also inhibits the absorption of β-sitosterol andcampesterol, which can be seen as a decrease in their concentrations.

Respectively, the changes caused by stanol additions in the total andLDL serum cholesterol concentrations and in cholesterol absorption werealso measured and these changes are shown in Table 2. The control groupconsumed ordinary rapeseed oil without stanol additions.

TABLE 2 Effect of rapeseed oil and β-sitostanol ester dissolved in it onthe absorption of cholesterol. Cholesterol Absorption at theIntervention Period Group (mg/d) Beginning End Change % Control RapeseedOil Rapeseed Oil +3.4 29.4 30.4   β-sitostanol Rapeseed Oil RapeseedOil + −27.4 ester (895)¹ 29.2 β-sitostanol ester 21.2^(xt) ^(x)= Changeis significant, p. < 0.05. ^(t)= Change is significant as compared withthe change in the control group, p 0.05. ¹= Amount in free stanol.Cholesterol absorption was effectively reduced by a β-sitostanol fattyacid ester mixture (27.4%) even if the stanol intake was relatively low,895 mg/day. The cholesterol absorption of the control group did notchange. The action of free β-sitostanol and a β-sitostanol fatty acidester mixture on the cholesterol concentration in serum, as comparedwith the control group, is seen in Table 3:

TABLE 3 Effect in serum of β-sitostanol added to rapeseed oil oncholesterol levels. Stanol Added to Change (%) Caused by the Addition¹Rapeseed Oil (mg/d) Total Cholesterol LDL Cholesterol β-sitosanol (895)−2.1 −6.4 β-sitostanol ester −9.5^(xt) −11.6^(t) (3150) ¹= Change hasbeen corrected by the % change in the control group which had receivedrapeseed oil. ^(x)= Change is significant, p < 0.05. ^(t)= Change issignificant as compared with the change in the control group, p < 0.05.A β-sitostanol fatty acid ester mixture decreased both totalcholesterols and LDL cholesterol more effectively than did rapeseed oiland free β-sitostanol. A β-sitostanol fatty acid ester mixture dissolvedin rapeseed oil (3.2 g of β-sitostanol/day) decreased total cholesterolby 9.5% more and LDL cholesterol by 11.6% more than did rapeseed oilalone. Respectively, the HDL/LDL cholesterol ratio rose significantly,from 0.32 to 0.52.

The studies carried out show clearly that by the addition ofβ-sitostanol fatty acid esters to, for example, food fats, significantadvantages can be achieved both in the national nutrition and in thetreatment of hypercholesterolemia, since 1) the mixture lowerscholesterol values in serum, 2) the mixture does not increase serumplant sterol concentrations, 3) the mixture can be used daily as a fatsubstitute in cooking normal food, even in large doses (0.2-20 g/d),whereby the intake of energy (calories) from fat decreases.

Lipid changes caused by β-sitostanol fatty acid esters, observed in thestudy, are to be considered highly significant from the viewpoint ofhealth. The significance of the results is emphasized by the possibilityof using the compound along-side food preparations as part of ordinarycooking and an ordinary diet. Research results show that during anintervention diet the β-sitostanol level in serum does not rise, andthat the levels of other plant sterols in the serum decrease. Thus thesaid β-sitostanol ester mixture is safe also for those few individualswho readily absorb all sterols or who have disturbances in sterolexcretion. Furthermore, daily fat substitution decreases an individual'senergy supply, since the effective β-sitostanol compound is notabsorbed, i.e., it acts as a non-energy producing part of fat. There isno evidence of the said β-sitostanol ester mixture hampering theabsorption of lipid-soluble vitamins or the vitamin levels in serum.

The uses of a β-sitostanol fatty acid ester mixture as a part of variousfats and oils in fat-containing products are wide, since the physicalproperties of the mixture can be modified easily by altering the fattyacid composition of the mixture. In addition to this, the fatty acidcomposition of the β-sitostanol fatty acid ester mixture can be selectedso as to contain large amounts of monoenes and polyenes, whereby itsefficacy in lowering the cholesterol levels in serum are enhanced.

Since the β-sitostanol fatty acid ester mixture is prepared using rawmaterials belonging to normal food and production processes generallyused in the food industry, there are no obstacles to the production anduse of the compound. For example, but without limitation, the sitostanolester is useful in such foods as cooking oils, margarines, butter,mayonnaise, salad dressings, and shortenings.

To test the long-term tolerability and hypocholesterolemic effect ofβ-sitostanol esters, a one year long randomized double blind study wasperformed. The ester was administered as a β-sitostanol ester richmargarine and the effect of this margarine was studied in a randomlyselected mildly hypercholesterolemic population sample of 150 persons.

The participants were recruited from the random population of about 1560previously studied for serum total and HDL cholesterol andtriglycerides. The major selection criteria were as follows: Serum totalcholesterol≧5.58 mmol/l, triglycerides<3 mmol/liter, age 25-65 years,BMI<30, stable medication for hypertension, diabetes or coronary heartdisease and absent renal, alcohol, liver or thyroid problems. Malesaccounted for 42% of the population.

After a fasting blood sample the participants replaced 24 g/day of theirdietary fat for 6 weeks through a rapeseed oil based margarine. Themajor fatty acid of this margarine was: C16:0=16.7%, C18:1=47.3%, andC18:3=8.9%. The total amount of trans fatty acids was 0.5%. Themargarine was distributed as 8 gram portion packs to be used on a sliceof bread with breakfast, lunch, and dinner. The actual amounts ofmargarine consumed per portion pack ranged, as measured during differenttimes of the study, from 7.3 to 7.7 grams.

At the end of the 6-week period, the persons were randomized to eithercontinue on their rapeseed oil based margarine without addedβ-sitostanol ester (control group: n−50) for 12 months or to change tothe same margarine with β-sitostanol ester added (sitostanol estergroup: n=100). The daily intake of β-sitostanol was intended to be 3grams. After a 6-month period the sitostanol ester group wasrerandomized to continue either on a daily intake of β-sitostanol ofeither 3 gram (n=50) or 2 gram (n=50). The persons were not informedabout this change which was achieved by changing the content ofβ-sitostanol ester in the margarine. The amount of margarine wasunchanged throughout the study. After the actual study period of 12months with different margarines the people returned to their regularhome diets. Blood samplings were made twice at 0 (the end of the 6-weekcontrol margarine period), 12 (end treatment) and 14 (post-treatment)months, a week apart and once at 1, 5, 3, 6, and 9 months. Thepost-treatment blood samples were taken to record the lipid values ofthe home diet after the margarine studies.

Results

Body weights of the 3 groups were similar at the baseline and remainedconstant during the study. The daily consumption of margarines wassimilar after the first (19.2-20.0 g/day) and second (19.0-19.2 g/day)randomization in the different groups. The margarines were welltolerated and the use of the test margarines on bread was similar in thedifferent groups. On average, each person consumed 86% of the deliveredmargarine (22.5 g/day). Measurements of β-sitostanol in the margarineand the actual intake of margarine showed that the mean daily intake ofβ-sitostanol was 2.6 g and 1.8 g in the sitostanol ester groups.

No side effects were reported. Data obtained showed that α-tocopherolcontent in serum in the 50 participants on margarine withoutβ-sitostanol ester was 17.1±1.8 mg/dl after the initial 6-week periodwith control margarine and 18.7±1.5 mg/dl at 12 months. The respectivevalues in the β-sitostanol ester groups were 15.1±1.0 (before the use ofβ-sitostanol ester containing margarine) and 13.8±1.4 mg/dl (withβ-sitostanol ester). Thus, the changes in the two groups were notsignificantly different from each other. The lowering of LDL cholesterol(major transporter lipoprotein for α-tocopherol) by 15% could clearlyexplain the nonsignificant decrease in α-tocopherol in the β-sitostanolester groups. It can be postulated that the α-tocopherol concentrationof the remaining LDL particles is even higher after the use ofβ-sitostanol ester.

Serum Lipids

The serum lipid values obtained is given in Table 4. The baseline lipidlevels were similar in the 3 groups. The use of the control margarinefor the additional 12 months after the 6-week initial period did notchange the serum total, LDL, HDL cholesterol or triglyceridesconcentrations, but was followed by a significant increase in the serumcontents at the home diet.

TABLE 4 Serum lipids and lipoproteins during consumption of margarinewithout and with β-sitostanol fatty acid ester. Time Cholesterol, mmol/lTriglycerides Months Total LDL HDL mmol/l Margarine Group I (n = 50) −1½6.12 ± 0.12 4.10 ± 0.11 1.39 ± 0.05 1.40 ± 0.09  0 6.12 ± 0.10 4.13 ±0.10 1.37 ± 0.05 1.37 ± 0.08  6 6.06 ± 0.10 4.09 ± 0.11 1.34 ± 0.05 1.40± 0.09 12 6.11 ± 0.10 4.08 ± 0.10 1.37 ± 0.05 1.44 ± —/—0 14 6.29 ± 0.144.26 ± 0.11 1.36 ± 0.06 1.50 ± 0.12 Sitostanol (2.6 g) Margarine GroupII (n = 50) −1½ 6.08 ± 0.11 4.12 ± 0.10 1.37 ± 0.05 1.29 ± 0.08  0 6.07± 0.11 4.14 ± 0.11 1.36 ± 0.05 1.27 ± 0.07  6 5.53 ± 0.10* 3.58 ± 0.18*1.33 ± 0.04 1.17 ± 0.05 12 5.42 ± 0.10* 3.51 ± 0.09* 1.36 ± 0.03 1.23 ±0.06 14 6.10 ± 0.12 4.12 ± 0.12 1.34 ± 0.04 1.43 ± 0.11 Sitostanol (1.7g) Margarine Group III (n = 50) −1½ 6.07 ± 0.10 4.02 ± 0.9 1.49 ± 0.041.24 ± 0.08  0 6.00 ± 0.10 3.96 ± 0.09 1.47 ± 0.05 1.24 ± 0.06  6 5.46 ±0.18* 3.45 ± 0.09* 1.44 ± 0.05 1.26 ± 0.09 12 5.53 ± 0.10* 3.45 ± 0.08*1.50 ± 0.04 1.28 ± 0.07 14 6.08 ± 0.12 3.96 ± 0.10 1.50 ± 0.05 1.38 ±0.09 All three groups consumed home diet at −1½ and 14 months, margarinefrom −1½ to 0 in all groups and in Group I further to 12 months. GroupII consumed sitostanol 2.6 g/day from 0 to 12 months and Group III 2.5g/day from 0 to 6 months and 1.8 g/day from 6 to 12 months.*Significantly different (p < 0.001) from 0 value or respective Group Ivalue.

The Figure shows serum cholesterol levels of the three groups ofparticipants before, during, and after twelve months eating of margarinewith and without sitostanol fatty acid ester.

Addition of β-sitostanol ester (β-sitostanol 2.6 g/day) to the margarinedecreased serum total cholesterol by 8.7% and LDL cholesterol by 13.5%at 6 months, and by 10.7% and 15.2% by 12 months. The respectivecontrol-related values are 8.7, 10.6, 12.7, and 14.2%. The reduction ofβ-sitostanol intake to 1.8 g/day at 6 months showed a small decrease inthe reduction of serum total cholesterol levels so that a statisticaldifference could be seen between 6 and 12 months in the cholesterolcurves of the two groups.

After the persons resumed their normal diet, the values returned back tothe initial level. Serum HDL cholesterol and triglyceride levels werenot affected by the use of β-sitostanol ester. This means, that the HDLcholesterol/total cholesterol ratio or the HDL cholesterol/LDLcholesterol ratio were increased by β-sitostanol.

The data obtained shows that a simply modified dietary fat i.e., aregular margarine fortified with fat soluble β-sitostanol estersdecreases serum total and LDL cholesterol concentrations by about 11-15%in the mildly hypercholesterolemic part of a randomly selectedpopulation sample. The findings of this study suggests favorable effectsof long-term use of β-sitostanol ester margarine to substitute a part ofthe normal dietary fat intake. β-sitostanol itself is unabsorbable, doesnot appear to interfere detectably with the fat soluble vitamins and istasteless to the extent that it can be consumed in sufficiently largeamounts to cause a moderate cholesterol lowering effect in a relativelysmall part of dietary fat. Thus, the partial substitution of dietary fatby β-sitostanol ester margarine is suitable not only for thepopulation-wide strategy of lowering serum cholesterol, but also as partof a strategy to reduce hypercholesterolemia in the high-riskpopulation. Thus, this kind of fat preparation is recommended for thegeneral prevention of coronary heart disease and other atheromatousdiseases in the population. Based on this study the preferred daily dosefor β-sitostanol ester is 2.5-6.5 grams.

Example 1

A β-sitostanol ester mixture was prepared on a pilot scale. 6 kg ofβ-sitostanol which had been dried overnight at 60° C. was esterifiedwith 8.6 kg of a rapeseed oil methyl ester mixture. The esterificationwas carried out as follows:

A mixture of β-sitostanol and rapeseed oil fatty acid methyl ester washeated in a reaction vessel at 90°-120° C. and under a vacuum of 5-15mmHg. The drying was continued for an hour, 12 g of Na ethylate wasadded, and the reaction was continued for approximately 2 hours. Thecatalyst was destroyed by adding water to the mixture. After phaseseparation, the oil phase was dried under a vacuum.

A conversion of 98% was achieved in the reaction. The obtained estermixture can be used as such as an additive in fats.

Instead of a mixture of rapeseed oil fatty acid esters it is possible touse in the reaction a methyl ester or a methyl ester mixture of thefatty acids of any vegetable oil, especially of fatty acids whichcontain approximately 2-22 carbon atoms.

Example 2

Before the steam blowing of rapeseed oil, β-sitostanol ester mixtureprepared in Example 1 was added, at 3, 6, and 13% by weight, to therapeseed oil. Mayonnaise containing the said fat mixtures at 65% wereprepared.

Mayonnaise:

% fat mixture 65.0 thickening agent 2.0 salt 1.0 sugar 3.0 vinegar (10wt. %) 3.0 mustard 2.0 water 24.0 Total 100.0

The mayonnaise was prepared by homogenization by a known manner using aKoruma homogenizer.

There were no problems in the preparation of the mayonnaise, and theirproperties tested by sense perception did not differ from those ofconventional mayonnaise.

Example 3

Before the steam blowing of oil, β-sitostanol ester mixture prepared inExample 1 was added, at 3 and 6% by weight, to the rapeseed oil.

The rapeseed oil to which the ester mixtures had been added remainedclear at room temperature, and no permanent turbidity was observed in itwhen it was stored at refrigerator temperatures.

Example 4

Other oils, such as sunflower, soybean, olive and corn oil, can also beused as the oil in the products according to Examples 2 and 3.

Example 5

β-sitostanol ester mixture prepared in Example 1 was added, at 10 and20% by weight, to the fatty part of a conventional soft margarine(composition: partly hardened soybean oil 35%, coconut oil 5%, rapeseedoil 60%) before the steam blowing of the fat mixture.

The DP (dropping point) and values of the mixtures were analyzed:

-   -   1) the mixture as such    -   2) the mixture+ester mixture at 10%    -   3) the mixture+ester mixture at 20%

Mixture NMR values (%) DP (° C.) 10° C. 20° C. 30° C. 35° C. 40° C. 45°C. 1) 31.9 24.2 11.6 2.7 0.7 0.0 0.0 2) 30.4 21.4 10.0 1.8 0.2 0.0 0.03) 29.6 25.4 9.2 2.0 0.6 0.0 0.0A margarine which contained fat 80% was prepared by a generally knownmethod. The physical and sense perceivable properties of the margarinecorresponded to those of conventional margarines.

Example 6

A five weeks double blind study was performed to show serum cholesterollowering effects of the margarine containing 12.5% β-sitostanol estersand to find out possible effects on the taste and palatability of theproduct.

A group of 24 persons was randomized based on their blood serum lipidvalues into two groups, a stanol ester group and a control group. Thestanol ester group (n=12) consumed 3 portion packs of 8 g β-sitostanolester enrichment. All persons knew that half of them got β-sitostanolester enriched margarine and they were instructed to use the margarineon bread. The effect of the β-sitostanol esters on blood serum lipidvalues were typical, with a reduction in total cholesterol and LDLcholesterol of 9% and 13%, respectively. At the end of the diet periodeach person was asked whether his/her margarine was enriched withβ-sitostanol esters. The following answers concerning the margarine wasobtained:

TABLE 5 β-sitostanol Ester Control Group Group Yes 5 2 No 1 4 I can'tsay 6 6

As can be seen only 1 of 12 in the control group and 2 of 12 in theβ-sitostanol ester group gave the correct answer. This data clearlyshows that incorporation of β-sitostanol ester in margarine even at ashigh levels as 12.5% do not cause any appreciable changes to the tasteor other properties of the product.

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1. A method for reducing the cholesterol level in blood serum of asubject, comprising orally administering to the subject a blood serumcholesterol level reducing effective amount of at least one 5α-saturatedsterol fatty acid ester.
 2. The method of claim 1, wherein the at leastone 5α-saturated sterol fatty acid ester comprises a sitostanol fattyacid ester.
 3. The method of claim 1, wherein the at least one5α-saturated sterol fatty acid ester is administered as a component of afood composition.
 4. The method of claim 1, wherein the at least one5α-saturated sterol fatty acid ester is administered as a supplement toa food composition.
 5. The method of claim 1, wherein the at least one5α-saturated sterol fatty acid ester is administered at a dose ofbetween about 0.2 and about 20 grams per day.
 6. The method of claim 1,wherein the at least one 5α-saturated sterol fatty acid ester isadministered at a dose of between about 2.5 and about 6.5 grams per day.7. The method of claim 1, wherein the at least one 5α-saturated sterolfatty acid ester is administered at a dose of between about 895 andabout 3150 milligrams per day, calculated as free 5α-saturated sterol.8. The method of claim 1, wherein the fatty acid part of the estercontains about 2-22 carbon atoms.
 9. The method of claim 1, wherein theat least one 5α-saturated sterol fatty acid ester is produced by aninteresterifying step which is carried out in a food-grade process. 10.The method of claim 2, wherein the at least one 5α-saturated sterolfatty acid ester is administered as a component of a food composition.11. The method of claim 2, wherein the at least one 5α-saturated sterolfatty acid ester is administered as a supplement to a food composition.12. The method of claim 2, wherein the at least one 5α-saturated sterolfatty acid ester is administered at a dose of between about 0.2 andabout 20 grams per day.
 13. The method of claim 2, wherein the at leastone 5α-saturated sterol fatty acid ester is administered at a dose ofbetween about 2.5 and about 6.5 grams per day.
 14. The method of claim2, wherein the at least one 5α-saturated sterol fatty acid ester isadministered at a dose of between about 895 and about 3150 milligramsper day, calculated as free 5α-saturated sterol.
 15. The method of claim2, wherein the fatty acid part of the ester contains about 2-22 carbonatoms.
 16. The method of claim 2, wherein the at least one 5α-saturatedsterol fatty acid ester is produced by an interesterifying step which iscarried out in a food-grade process.
 17. A method for increasing theHDL/LDL cholesterol ratio in a subject, comprising orally administeringto the subject an HDL/LDL cholesterol ratio increasing effective amountof at least one 5α-saturated sterol fatty acid ester.
 18. The method ofclaim 17, wherein the at least one 5α-saturated sterol fatty acid estercomprises a sitostanol fatty acid ester.
 19. The method of claim 17,wherein the at least one 5α-saturated sterol fatty acid ester isadministered as a component of a food composition.
 20. The method ofclaim 17, wherein the at least one 5α-saturated sterol fatty acid esteris administered as a supplement to a food composition.
 21. The method ofclaim 17, wherein the at least one 5α-saturated sterol fatty acid esteris administered at a dose of between about 0.2 and about 20 grams perday.
 22. The method of claim 17, wherein the at least one 5α-saturatedsterol fatty acid ester is administered at a dose of between about 2.5and about 6.5 grams per day.
 23. The method of claim 17, wherein the atleast one 5α-saturated sterol fatty acid ester is administered at a doseof between about 895 and about 3150 milligrams per day, calculated asfree 5α-saturated sterol.
 24. The method of claim 17, wherein the fattyacid part of the ester contains about 2-22 carbon atoms.
 25. The methodof claim 17, wherein the at least one 5α-saturated sterol fatty acidester is produced by an interesterifying step which is carried out in afood-grade process.
 26. The method of claim 18, wherein the at least one5α-saturated sterol fatty acid ester is administered as a component of afood composition.
 27. The method of claim 18, wherein the at least one5α-saturated sterol fatty acid ester is administered as a supplement toa food composition.
 28. The method of claim 18, wherein the at least one5α-saturated sterol fatty acid ester is administered at a dose ofbetween about 0.2 and about 20 grams per day.
 29. The method of claim18, wherein the at least one 5α-saturated sterol fatty acid ester isadministered at a dose of between about 2.5 and about 6.5 grams per day.30. The method of claim 18, wherein the at least one 5α-saturated sterolfatty acid ester is administered at a dose of between about 895 andabout 3150 milligrams per day, calculated as free 5α-saturated sterol.31. The method of claim 18, wherein the fatty acid part of the estercontains about 2-22 carbon atoms.
 32. The method of claim 18, whereinthe at least one 5α-saturated sterol fatty acid ester is produced by aninteresterifying step which is carried out in a food-grade process.